Our research objective is to understand the biochemical events involved in apoptosis. Apoptosis is an important mode of cell death relevant for many developmental, immunologic, and pathologic processes. Like cell division and differentiation, apoptosis is complex but some of the important steps are shared. Unraveling the signaling events for diverse apoptotic stimuli should lead to a better understanding of diseases in which cell death is dysregulated (cancer, AIDS, degenerative disorders) as well as the normal regulation of immune responses. Most models for apoptosis suggest that various stimuli, with unique cellular targets, induce a common cell death signal that triggers the controlled destruction of the cell. These later events appear to involve the activation of caspases (ICE-family proteases). We have used a transient transfection death assay so that the effect of various gene products on apoptosis can be readily assessed. Previous work examined the role of Bcl-2 proteins and caspases in the regulation of apoptosis. Overexpression of Bcl-2 family members in T cell hybridomas efficiently blocked killing induced by cytotoxic agents but not anti-Fas whereas CrmA, an ICE-like protease inhibitor, showed the opposite pattern. Importantly, these findings have been confirmed in normal cells by transiently transfecting T cell blasts. These results suggest that there are at least two apoptotic signaling pathways that show differential sensitivity to Bcl-2 and caspase inhibitors. We are investigating the basis for the stimulus- and cell type-dependent activity of Bcl-2 and caspase inhibitors. Current projects; 1) Examine the role of specific caspases during apoptosis: caspases have been cloned and epitope tagged so that proteolytic activation by cell death stimuli can be monitored and the effect of Bcl-2 and caspase inhibitors assessed, 2) Chimeric caspase receptors as probes of Fas signaling and caspase biology: chimeric receptors are being characterized that show inducible caspase activation and apoptosis, 3) Identify novel activators, regulators, and substrates of caspases by yeast two hybrid analysis: caspase binding proteins have been identified and are being assessed for biochemical interaction and functional activity.